Real Time PCR

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Real Time PCR
Raheel Qamar, PhD. Professor Chairman Dept. of
Biosciences COMSATS Institute of Information
TechnologyIslamabad http//www.raheelqamar.com
Raheel Qamar, PhD. Research PCR Lab
Director Shifa College of MedicineIslamabad http
//www.raheelqamar.com
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Real-Time PCR

• Theory
• Chemistry
• Non-specific DNA binding dye
• Specific Hybridization Probe
• Dual-labeled FRET Pairs
• Advantages Disadvantages

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The Applied Biosystems Real Time PCR
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Smart Cycler by Cepheid
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iCycler iQ - Real Time PCR Detection System
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Real-Time PCR

• Through the use of fluorescent molecules,
  real-time PCR has the ability to directly measure
  the reaction while amplification is taking place.

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Real-Time PCR

• The fluorescent molecules can be
• Non-specific DNA binding dyes
• SYBR Green I
• Ethidium Bromide
• Specific Hybridization Probes
• Dual-labeled FRET pairs
• TaqManTM
• Pulsar LightCycler
• Molecular Beacons
• ScorpionsTM
• Amplifluor Direct

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Cycles and number of copies
No of copies
Cycles
No of copies
Cycles
No of copies
Cycles
1 2 4 8 16 32 64 128 256 512 1024
1024 2048 4096 8192 16384 32768 65536 131072 26214
4 524288 1,048,576
1,048,576 1,073,741,824 1,099,511,627,776
11 12 13 14 15 16 17 18 19 20
30 40
1 2 3 4 5 6 7 8 9 10
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Reality vs. Theory
Amplification is exponential, but the exponential
increase is limited

• A linear increase follows exponential
• Eventually plateaus

Real-Time PCR allows us to see the exponential
phase so we can figure out how much we started
with.
Cycle
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End Point Measurements 96 replicates of the
identical reaction have very different final
amounts of fluorescence
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End-point vs. Real-Time results
Lockey et al. (1998) Biotechniques 24744-6
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Threshold Cycle (Ct)
The point at which the fluorescence rises
appreciably above background
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Threshold Cycle, Ct

• Correlates strongly with the starting copy number
• If you have high DNA you get to Ct cycle
  earlier
• If you have low DNA you reach Ct cycle later
• Is linear with the log of starting copy number
  over 6 or more orders

Which one has the most?
The least?
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HCV Real Time PCR Quantification
HCV Standards
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HBV Real Time PCR Quantification
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Threshold Cycle, Ct vs initial copy number
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HCV Real Time PCR Quantification
HCV samples standard curve
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Borderline Samples
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Results of Retesting of Borderline Samples

• 69 borderline samples
• 27 (39) became positive upon retesting
• 27 (39) became negative upon retesting
• 6 (9) remained borderline upon retesting
• 9 (13) did not come back for retesting

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Real Time PCR run on Agarose gel
14,700
678,000
41,300
78
62
57
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What is Real Time PCR?

• Monitoring of an amplification reaction in
  progress.
• HOW?
• By tracking the amplification via an increase in
  fluorescent light

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Advantages of Real Time PCR
Data evaluated rapidlyINCREASING Throughput
No Post-PCR processingREDUCING Contamination
Stop reactions during faulty runs-SAVE Time
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Applications of Quantitative PCR

• Viral Particle concentration - Viral Load
• mRNA expression levels - Gene Therapy Efficacy,
  Drug Development, Disease State Monitoring
• Gene Amplification - Cancer Research
• Melting Temperature Behaviour - Mutation
  Detection in Heterozygocity (Allelic
  Discrimination)
• End Point Detection - Qualitative, screening of
  known mutations (SNP Analysis)

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What are the options for Real Time PCR?

• Chemistry strategies to measure Real Time PCR
  are
• Intercalating Dyes, e.g. Ethidium Bromide and
  SYBR Green
• Hybridization Probes - offer probe specificity

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Detection Strategies - DNA Binding Dyes

• DNA binding dyes are inexpensive compared to
  hybridization probes.
• A dye based strategy allows one to get a general
  confirmation of amplification.
• Higuchi demonstrated the key principle of
  real-time PCR using Ethidium Bromide -
• EtBr fluoresces 25 times more brightly when bound
  to dsDNA
• SYBR Green I, a more sensitive DNA binding dye,
  is an even more powerful approach
• SYBR Green I fluoresces 200 times more brightly
  when bound to dsDNA

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DNA Binding Dyes

• Prepare reaction mixture

d.NTPs
Primers
DNA binding dyes
Thermal Stable DNA Polymerase
Reaction Tube
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DNA Binding Dyes

• Denaturation

l
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• DNA Binding Dyes
• Annealing and extension

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DNA Binding Dyes

• Advantages
• Cheap
• Quick
• Easy
• Disadvantages
• Less specific
• Significant optimisation required to minimise
  primer-dimer formation and amplification of
  non-specific product

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Hybridization Probes

• Hybridization Probe Strategies
• Dual-labeled FRET probes
• Cleavage Based Assay - TaqManä Assays
• Displaceable Probe Assays
• Molecular Beacons
• Black Hole Scorpions

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TaqMan

• Fluorescein is excited at 488nm and emits light
  around 520nm
• The 520nm light excites the TAMRA which emits
  light around 570nm

TaqMan Probe
Taq Polymerase
- Reporter emitted light increases. - Cleavage
of probe occurs when probe has found target.
Reporter
Quencher
- increase in reporter emission represents
increase in the amount of amplification product
Fluorescent Tag
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Cleavage-based assay TaqMan
d.NTPs
Primers
Add Master Mix and Sample
Thermal Stable DNA Polymerase
Reaction Tube
Denaturation
l
Annealing
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Cleavage-based assay TaqMan
5
3
Extension Step
1. Strand Displacement
2. Cleavage
3. Polymerization Complete
l
4. Detection
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Choosing a Flourophore Quencher
Quenchers
Fluorophores
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Features of Real Time PCR

• Sensitive good detection capability at low
  concentrations
• Dynamic Range good detection of wide range of
  sample concentrations
• Accurate relationship between fluorescence and
  PCR product produced
• Multiplexing

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TaqMan
Advantages

• Amplification in signal due to large no of
  reporters generated
• 1st cycle 1x reporters.
• 2nd cycle 3x reporters.
• 3rd cycle 7x reporters.
• 25th cycle 33,554,431x reporters.
• Predominant assay in Real Time PCR literature
• Many reagents available for assay targets.

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TaqMan
Disadvantages

• Quencher always fluoresces.
• Adds extra signal
• Reporter must have emission spectra distinct from
  quencher
• Requires that reporters emit light in the range
  that will excite the quencher
• Multiplexing limited.
• Probe design options limited.

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Molecular Beacons
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Molecular Beacons SNP Analysis
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Black Hole Scorpions
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Real-Time PCR

• Theory
• Chemistry
• Non-specific DNA binding dye
• SYBR Green I
• Specific Hybridization Probe
• TaqManTM
• Molecular Beacons
• Black Hole Scorpions
• Advantages Disadvantages